Toxicity and Metabolism Testing of Potential Therapeutic Drugs and Chemical Entities
Drug-induced liver toxicity is an important clinical problem, and several drugs have been withdrawn from the market because of their ability to cause rare but severe (even lethal) cases of hepatotoxicity. Induction of cytochrome P450 (CYP) and related drug-metabolizing enzymes (DMEs), including transporters, is a well-recognized cause of clinically significant drug-drug interactions, as well as a cause of loss of efficacy (pharmacokinetic tolerance) or auto-induction (the process whereby a drug induces its own hepatic metabolism). During drug development, in vitro assays can be used to avoid inducers, and characterize drug-drug interaction potential due to increased drug clearance by the liver. In vitro induction studies traditionally use primary hepatocyte cultures and enzyme activity with selected marker compounds.
CYPs are involved in the metabolism of drugs, primarily in the liver. For example, induction of CYP3A gene expression is caused by a variety of marketed drugs including rifampin, phenobarbital, clotrimazole, and dexamethasone and represents the basis for a number of common drug-drug interactions (Meunier et al., Expression and induction of CYP1A1/1A2, CYP2A6 and CYP3A4 in primary cultures of human hepatocytes: a 10-year follow-up, Xenobiotica 30(6): 589-607, 2000; Sahi et al., Effect of troglitazone on cytochrome P450 enzymes in primary cultures of human and rat hepatocytes, Xenobiotica 30(3): 273-284, 2000; Luo et al., CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes, Drug Metab. Dispos. 30(7): 795-804, 2002; Madan et al., Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes, Drug Metab. Dispos. 31(4): 421-431, 2003).
Guidelines for assessing enzyme induction in vitro have been outlined in Tucker et al. (Optimizing drug development: Strategies to assess drug metabolism/transporter interaction potential—toward a consensus, Pharmaceutic. Res. 18: 1071-1080, 2001) and Bjorsson et al. (The conduct of in vitro and in vivo drug-drug interaction studies: A PhRMA perspective, J. Clin. Pharmacol. 43: 443-469, 2003). These two “consensus reports” identify primary cultures of human hepatocytes as the method of choice—the gold standard—for assessing the enzyme-inducing potential of chemical entities and drug candidates. This in vitro approach, based on a human-derived test system, is superior to an in vivo approach based on tests in laboratory animals because drugs are known to cause enzyme induction in a species-specific manner. For example, the two prototypical inducers, namely omeprazole and rifampin, are efficacious inducers of human CYP1A2 and CYP3A4 and yet they do not induce the corresponding enzymes in rats or mice.
Human hepatocytes play several key roles in preclinical drug development. They can be used to assess the effects of drug candidates on the liver in a clinically meaningful manner (e.g., the induction and cellular toxicity) and, conversely, they can be used to assess the effects of the liver on chemical entities (e.g., drug metabolism and species comparisons). Primary cultures of human hepatocytes have the distinct advantage of exhibiting species-specific induction of CYP isoforms; however, the utility of cryopreserved or plated primary human hepatocytes is restricted by the limited and erratic supply of human liver and by significant inter-individual differences in the expression of DMEs and responses to toxicants.
Cell lines of tumorigenic origin, such as HepG2 and H4IIE, are routinely used for comparison of the in vitro toxicity of candidate compounds. Such cells are unlikely to retain many or most of the factors that predict cell-specific toxicity in vivo. For instance, most tumor-derived cells are not highly differentiated; they rapidly proliferate in culture, which requires enormous energy (ATP consumption) and which may increase their sensitivity to cellular insult compared to non-proliferative cells.
Thus, there is a need for a nontumorigenic immortalized human hepatocyte cell line that retains the properties of a normal human hepatocyte, namely metabolic and transporter function, while offering the distinct advantages of reproducibility and unlimited availability.
Therapeutic Plasma Proteins
There is a great demand for therapeutic plasma proteins (TPPs), such as albumin, α-1 antitrypsin (AAT), blood clotting factors VIII and IX, and inter-α-inhibitor proteins (IαIp). The production of TPPs by cell-based systems would avoid the hazards of blood-derived products, such as potential contamination with viruses or other pathogens.
Currently, the majority of proteins that have been approved for clinical and therapeutic use are mass-produced by recombinant protein technology. Although these products have been proven safe and effective, not all behave identically to their native counterparts. For example, recombinant factors (rF) VIII and IX are more rapidly cleared following infusion than their plasma derived counterparts. Shapiro, A., E. Berntorp, and M. Morfini, Incremental recovery assessment and effects of weight and age in previously untreated patients treated with recombinant factor IX. Blood, 2000. 96 (suppl 1): p. 265a. Recent findings suggest that this is the result of incomplete or inappropriate post-translational modification.
Hemophilia A (Factor VIII deficiency) occurs in 1 in 5,000 to 10,000 males in the United States. In contrast, the incidence of hemophilia B (Factor IX deficiency) is 0.25 in 10,000 males. Currently, plasma-derived and recombinant Factor VIII and IX concentrates are used for the lifetime treatment of hemophilia. It is estimated that three-quarters of the worldwide hemophilia population receive little or no treatment due to a shortage of these TPPs. Thus, there is a clear need for fully functional, fully native blood-clotting factors that overcome the shortcomings of recombinant or blood-derived TPPs.
α-1-antitrypsin (AAT) is a human blood protein. Severe AAT deficiency (hereditary emphysema) is thought to affect around 150,000-200,000 individuals in Europe and the United States. Many respiratory diseases including AAT congenital deficiency, cystic fibrosis and chronic obstructive pulmonary disease are characterized by an imbalance of AAT and elastase in the lung. Administration of supplemental AAT is clinically effective at alleviating the deleterious effects to the lung that occur in these diseases.
Currently, there is only one plasma-derived AAT licensed in the United States, which has been in very limited supply. Thus, there is a clear need for a fully functional, fully native AAT that can overcome the shortcomings of recombinant or blood-derived TPPs.
Sepsis, a disease characterized by an overwhelming systemic response to infection, can strike anyone and can be triggered by events such as pneumonia, trauma, surgery and burns, or by conditions such as cancer or AIDS. In the United States, sepsis is the leading cause of death in the noncardiac intensive care unit and the 11th leading cause of death overall. Currently, treatment for sepsis is limited to attempts to manage the underlying infection and supportive therapy if the infection leads to organ dysfunction. Despite intensive medical care, up to 50% of patients still die from this illness.
Inter-α-inhibitor proteins (IαIp) are natural serine protease inhibitors found in relatively high concentration in plasma that play roles in inflammation, wound healing and cancer metastasis. Bost, F., M. Diarra-Mehrpour, and J. P. Martin, Inter-alpha-trypsin inhibitor proteoglycan family—a group of proteins binding and stabilizing the extracellular matrix. Eur J Biochem, 1998. 252: p. 339-346. IαIp is believed to have a predictive value in septic patients. Lim, Y. P., et al., Inter-trypsin inhibitor: decreased plasma levels in septic patients and its beneficial effects in an experimental sepsis model. Shock, 2000. 13 (Suppl.): p. 161. In-vivo animal studies using a sepsis rat model have shown that administration of IαIp dramatically improved survival rates. Yang S, et al., Administration of human inter-alpha-inhibitors maintains hemodynamic stability and improves survival during sepsis. Crit Care Med. March 2002; 30(3):617-22. The results strongly support the therapeutic potential of IαIp in the management of severe sepsis. Yet, there is no ready supply of IαIp for administration to septic patients. Thus, there is a clear need for fully functional, fully native IαIp to overcome the shortcomings of recombinant or blood-derived TPPs.
There are a number of patents and publications that describe immortalized cell lines: U.S. Pat. No. 6,107,043 (Jauregui); U.S. Pat. No. 5,665,589 (Harris); U.S. Patent App. No. 2002/0045262 A1 (Prachumsri);.and International publication No. WO 99/55853 (Namba). However, to date, among other things, the prior art cell lines do not provide a means to safely, effectively, and cost efficiently perform the protein post-translational modifications, such as glycosylation, that are critical in the production of functional therapeutic plasma proteins; produce simultaneously multiple therapeutic plasma proteins, especially factor VIII protein or factor IX; and maintain the continuous expression of active levels of cytochrome P450 enzyme in a serum-free media. Thus there is a need for a nontumorigenic immortalized human hepatocyte cell line that retains the properties of a normal human hepatocyte, and can be used to produce properly processed therapeutic plasma proteins.